The present invention relates to crystalline farnesyl protein transferase (FPT) and FPT in complex with substrates or inhibitors. This invention also relates to methods of using the structure coordinates of FPT to solve the structure of similar or homologous proteins or protein substrate or inhibitor complexes.
The biological significance of the Ras oncogene, and the role of both Ras and the enzyme known as farnesyl protein transferase (xe2x80x9cFPTxe2x80x9d) in the conversion of normal cells to cancer cells, are described in PCT International Publication Nos. WO95/00497 and WO95/10516. To undergo transforming potential, the precursor of the Ras oncoprotein must undergo farnesylation of the cysteine residue located in a carboxyl-terminal tetrapeptide. Farnesyl protein transferase catalyzes this modification. Inhibitors of this enzyme have therefore been suggested as anticancer agents for tumors in which RAS contributes to transformation.
Drug discovery efforts directed toward FPT inhibitors have been hampered by the lack of adequate structural information about FPT and its complex with substrates and inhibitors. The structure of FPT was first determined at Duke University using a crystalline form where the active site was blocked by the carboxy terminus of an adjacent molecule in the crystalline lattice. Beese et al., 1997, Science 275:1800-1804. These crystals are not suited for drug discovery because, as reported therein, the active site is blocked by part of the crystal lattice. Another crystalline form of FPT was also reported in Dunten et al., 1998, Biochemistry 37(22):7907-7912. These crystals grow at pH 4.4 and are reported to only diffract to 2.8 xc3x85 resolution. However, the authors point out that there are no substrates or peptide inhibitors bound due to the low pH of the crystallization. Thus these crystals are not suitable for structure-based drug design.
Structural information from FPT crystalline complexes would provide valuable information in discovery of FPT inhibitors. This information could be used to design more potent, selective and metabolically stable FPT inhibitors for use as drugs against cancer.
Applicants have solved this problem by providing, for the first time, a crystallizable composition comprising a farnesyl protein transferase (FPT) complexed with molecules that mimic its natural substrates. The invention also provides crystals of FPT complexed with FPP or an FPP analog and a peptide or an inhibitor. (These complexes are referred to throughout as FPT:FPP/FPP analog:Peptide/Inhibitor complexes.) The invention also provides the structure coordinates of these complexes. Further provided are methods and reagents for soaking these crystalline complexes in the presence of an inhibitor, thereby efficiently forming a crystalline enzyme: inhibitor complex.
The invention also provides a method for determining at least a portion of the three-dimensional structure of molecules or molecular complexes which contain at least some structurally similar features to an FPT: substrate or FPT: inhibitor complex.